a. Introduction
The generation and decay of proteolytic enzymes in body fluids is a key element in processes as diverse as digestion, inflammation, blood coagulation and thrombosis. To give an example: Thrombin is an enzyme that is transiently present in clotting blood and that is the key enzyme of haemostasis and thrombosis. Disorders of the haemostatic thrombotic system (HTS) are pivotal in over half of all invalidating and lethal disease. Quantitatively the less important ones, haemophilia and lung embolism, come readily to mind. It is not so widely acknowledged that arterial thrombosis causes coronary infarction or that one out of ten of the elderly risk loss of brain function through clots obstructing brain arteries (embolisation on the basis of atrial fibrillation and carotid emboli), or that seriously ill patients may bleed to death because of disorders of the clotting system (victims of accidents and patients suffering from sepsis with fatal intravascular coagulation). It is insufficiently recognised that more people die from arterial thrombosis than from malignancies and more from venous thrombosis than from accidents. In view of such medical importance it is surprising to note that there is no valid function test of the HTS available to the clinician today.
In body fluids there exist several more physiologically important biochemical systems that act through activation and subsequent inactivation of proteolytic enzymes, such as, in blood, the coagulation system, the fibrinolytic system and the complement system, and in gastrointestinal juices the digestive enzymes. For the assessment of biological function of these systems it is important to be able to follow the course of such proteolytic activity as it develops after triggering in a sample of the body fluid ex vivo in time. Such function assessment is of paramount diagnostic importance because disturbances of such systems can lead to fatal diseases like coronary infarction, stroke or fatal bleeding (blood coagulation and fibrinolysis), generalised infections and autoimmune diseases (complement system) or disturbed absorption of food (gastrointestinal juices).
In haemostasis and thrombosis, clotting times are the best assessments available today and they are insensitive to mild haemostatic disorders (e.g. carriers of haemophilia, mild liver disease) or to increased coagulability that leads to increased thrombosis risk. Clotting tests often need to be adapted to a specific use. For example, the thromboplastin time (=Prothrombin time (PT), =Quick time) can be used for diagnosis of serious liver disease or treatment with anticoagulants but is not prolonged by haemophilia or heparin treatment. Much of the art and science of the clinical coagulation lab resides in knowing how to interpret the scattered information that can be obtained from clotting times of different types, platelet aggregation, bleeding time etc.
The insufficiency of the over-all tests is partly compensated by a great variety of sophisticated tests of single components of the clotting system, so many indeed that a judicious selection should be made in every special case, which is the other half of the specific knowledge of the clinical haemostasis lab.
b. Mechanism of Thrombin Generation
The mechanism of thrombin generation in blood plasma can be exemplified as follows. Tissue Factor (TF) is abundantly—but not exclusively—present in the vessel wall. When a blood vessel is damaged, the blood enters the tissues and the plasma protein factor VIIa (VIIa) can interact with TF. This triggers an extremely complicated set of interactions, between plasma proteins and blood platelets, which results in a transient burst of thrombin that remains limited in time and space, so that normally a wound stops bleeding but clotting is not propagated in the remainder of the body.
This mechanism can be shown to be so intricate, replete with positive and negative feedback reactions, that its action cannot be predicted from knowledge of its parts (irreducible complexity). Therefore, if one wants to assess the function of the haemostatic system the thrombin generation has to be investigated as it occurs in the body, or in an isolated part of the body, i.e. a sample of blood or platelet-rich plasma The interaction between blood platelets and plasma factor is of particular importance, the information to be obtained from platelet-poor plasma being essentially deficient. See, e.g., Béguin S., R. Kumar, I. Keularts, U. Seligsohn, B. C. Coller and H. C. Hemker, Fibrin-Dependent Platelet Procoagulant Activity Requires GPIb Receptors and Von Willebrand Factor, Blood (1999) 93:564-570; Béguin, S. and R. Kumar, supra (1997)].
An important fraction (≈30%) of all thrombin formed in clotting plasma is bound to the fibrin clot. Clot-bound thrombin does retain its thrombotic properties, it can clot fibrinogen, activate factors V, VIII and XI as well as platelets [Béguin, S. and R. Kumar, Thromb. Haemost. (1997) 78:590-594; Kumar, R., S. Béguin, and H. C. Hemker, Thromb. Haemost. (1994) 72:713-721, and (1995) 74:962-968]. It is only partly inhibited by antithrombin. Therefore, it is essential that fibrin is present when investigating the function of the coagulation system.
In order to assess the function of such a system for diagnostic purposes and for the safe use of antithrombotic drugs a variety of tests has been developed of single components of the clotting system, which will be further detailed below.
As stated before, the thrombin activity that generates at the site of a lesion is an important determinant of the extent of the haemostatic-thrombotic reaction that ensues. Most of the thrombin (>95%) generates after the moment of clotting, therefore the clotting time is not automatically a good indicator of thrombin activity. Thrombin activity in clotting blood is a transient phenomenon and therefore should be measured during the clotting process.
A typical course of thrombin formation in clotting blood or plasma, also designated as the thrombin generation curve, is shown in FIG. 1. After a period in which no observable thrombin is formed, the concentration steeply goes up, rises to a peak and then goes down again. The parameters are the lag time, the area under the curve (AUC), also designated as the endogenous thrombin potential (ETP; see below), the peak height, and the time it takes to reach the peak.
c. Related Prior Art
A thrombin generation curve as shown in FIG. 1 is classically obtained via determination of the thrombin content in small subsamples taken at short intervals from clotting blood or plasma. See, e.g., R. Biggs and R. G. Macfarlane, Human Blood Coagulation and its Disorders, Blackwell Scientific Publications, Oxford 1953; W. Seegers, Prothombin, Harvard University Press, Cambridge Mass. 1962. This method generally requires separate analysis of the subsamples and allows the determination of only 3-5 curves simultaneously by the continuous occupation of a skilled laboratory worker. It is so labor intensive as to preclude its application in clinical or pharmaceutical routine.
EP-A-0 420 332 (equivalent to U.S. Pat. No. 5,192,689) discloses a method to determine the amount of thrombin which has been present in a sample of either clotting blood or plasma by measuring the amount of product that is produced from an artificial substrate during coagulation. This amount is proportional to the area under the thrombin generation curve, designated as the endogenous thrombin potential, ETP. The method comprises adding a thrombin formation activator to a sample of either clotting blood or plasma together with a thrombin substrate, wherein the amount and also the kinetic properties of the thrombin substrate are chosen such that the amount of thrombin generated in the sample cannot completely consume said thrombin substrate, thereby to produce a conversion product, measuring the amount of said conversion product thus produced, and from this determining the endogenous thrombin potential in the sample. This ETP-method can be illustrated by the following reactions:

All reactions irreversible and therefore thrombin is only temporarily present in the reaction mixture. While thrombin is present, it participates in reaction 4, with the result that the degree of conversion of the substrate indicates the time for which, and the time to which, thrombin has catalyzed this reaction.
It is essential that the amount of substrate should not be exhausted before the thrombin disappears. For the amount of substrate converted to be an exact representation of the total amount of thrombin activity that developed, the reaction rate should be proportional to the concentration of thrombin at any instant in time. The essence of this ETP method is that the thrombin potential is determined as an end-point method without determination of the thrombin/time curve as such. In case the substrate would be short-measured, the end-point would be simply the maximum amount of product formed, and such figure has no meaning anyway.
Furthermore, the ETP-method is conducted in actual practice with a chromogenic substrate i.e. substrates with a chromophoric leaving group that is detected via optical density measurement. Fibrinogen, and consequently blood platelets, have to be removed from plasma because turbidity arising from the fibrinogen-fibrin conversion by thrombin makes further measurement impossible. Fibrinogen and platelets, however, are essential components of the clotting system that influence the course of thrombin formation. This puts a serious limit on the applicability of optical density as a detection method. Thus, assessment of the ETP in plasma containing platelets and/or fibrinogen would not be possible.
Continuous monitoring of thrombin concentration has been attempted through adding a suitable thrombin substrate to the clotting sample and monitoring the time course of appearance of the amidolytic split product. For example, a chromogenic substrate is used and the optical density is measured so as to monitor the development of p-nitro-aniline (Hemker H C, S. Wielders, H. Kessels, S. Béguin: Thromb Haemost. (1993) 70(4):617-24; Hemker H C, and S. Béguin: Thromb Haemost. (1995) 74(1):134-8). If the reaction velocity in such a test would be dependent upon thrombin concentration only and if the signal would be proportional to the amount of product, then the slope of the product curve would be proportional to the amount of thrombin present, so that the thrombin generation curve can be obtained from the first derivative of the product curve if the proportionality constant (Kc) is known.
In practice, however, the reaction velocity is not dependent upon the thrombin concentration only, the signal is not necessarily proportional to the amount of product and Kc is unknown. The reasons are the following:                A: Substrate consumption: The signal is not only dependent upon the activity of thrombin in the sample but also on the amount of substrate which, through the very enzyme activity itself, decreases in time. The effect can be attenuated by adding an excess of substrate but to a certain limit only. The substrate binds, reversibly, to the active centre of thrombin and thereby protects thrombin from inactivation by natural antithrombins. Abolishing the effect of substrate consumption to an acceptable degree is paid for by prolonging the experiment to last for about two hours. (The more substrate is added the more enzyme molecules are occupied and unavailable to the natural inactivation processes. This prolongs the duration of the experiment. At 1×Km the experiment is finished in 30 min, at 5 Km practical independence of substrate consumption is obtained but the experiment lasts 90 min). Also, at such concentrations of substrate, thrombin inhibition interferes with feedback reactions and it is no longer guaranteed that the natural process is measured. This is also the reason that, in a method meant to assess the area under the curve from the total amount of substrate converted, an excess of substrate has to be added such that extra antithrombin needs to be added in order to make the experiment practically possible (see EP-A-0 420 332, discussed above).        B: Changes in optical density occur through clotting of the plasma sample. The use of chromogenic substrates implies the removal of fibrinogen, and consequently blood platelets, that causes spurious increase of OD through scattering of light at the moment of clotting. Fibrinogen and platelets, however, are essential components of the clotting system that influence the course of thrombin formation (see above). This puts a serious limit on the applicability of optical density as a detection method. This problem can be circumvented by using a substrate that yields a fluorescent product [H. C. Hemker et al. The thrombogram: monitoring thrombin generation in platelet rich plasma. Thromb Haemost. 83:589-91 (2000)]. This, however, introduces the next problem:        C: In fluorescence measurements the signal is not linearly related to the amount of product. Notably the fluorescent signal is not linearly dependent upon the concentration of fluorescent product because fluorescent molecules absorb the light from other product molecules, the so called “inner filter effect”. With fluorescent products, increasing substrate concentrations to several times Km, as required for limiting the effect of substrate consumption automatically also increases the inner filter effect.        
Problem A is common to all continuous methods. Problem B can be circumvented by using a fluorogenic substrate but this introduces problem C.                D: Even if the problems A, B and C would not exist, the question remains of relating reaction velocity to thrombin concentration, i.e. determining the calibration constant Kc. This relation varies from experimental setup to experimental setup (e.g. is different in different fluorometers) and from sample to sample (e.g. due to color variations of the plasma). Addition of a known standard amount of thrombin to the sample is impossible because the enzyme added will disturb the physiological reactions. It is also impossible to add thrombin to a parallel non-clotting sample because it will be inactivated in the plasma.        
The present invention aims at obviating these drawbacks by providing a method relating to the determination of thrombin in a blood or plasma sample which is essentially different from the ETP-method outlined above, in that no end-point determination of the amount of product is made but rather the course of the thrombin concentration curve in real time is determined and provided as a continuous signal, thereby giving more valuable and accurate information regarding such parameters as lag time and peak height. The latter is more important for measuring subtle differences in the activity of the clotting mechanism as will be further outlined below. In other words, the new method does not provide a single value of the amount of thrombin that had been present in a sample as in the ETP-method, but rather provides the course of the thrombin concentration in real time that is transiently present in the sample.